U.S. patent application number 10/599942 was filed with the patent office on 2008-11-27 for diagnosis of inflammatory bowel diseases, more particularly ulcerative colitis.
This patent application is currently assigned to EUROIMMUN Medizinische Labordiagnostika AG. Invention is credited to Lars Komorowski, Swantje Mindorf, Winfried Stocker.
Application Number | 20080293625 10/599942 |
Document ID | / |
Family ID | 34964065 |
Filed Date | 2008-11-27 |
United States Patent
Application |
20080293625 |
Kind Code |
A1 |
Stocker; Winfried ; et
al. |
November 27, 2008 |
Diagnosis of Inflammatory Bowel Diseases, More Particularly
Ulcerative Colitis
Abstract
The present invention concerns goblet cell antigen, a method for
detecting antibodies directed against goblet cell antigen, a method
based thereon for diagnosis of inflammatory bowel diseases and a
kit for diagnosis of inflammatory bowel diseases, as well as
monoclonal antibodies directed against goblet cell antigen.
Inventors: |
Stocker; Winfried; (Gross
Gronau, DE) ; Mindorf; Swantje; (Burg, DE) ;
Komorowski; Lars; (Ratzeburg, DE) |
Correspondence
Address: |
Ballard Spahr Andrews & Ingersoll, LLP
SUITE 1000, 999 PEACHTREE STREET
ATLANTA
GA
30309-3915
US
|
Assignee: |
EUROIMMUN Medizinische
Labordiagnostika AG
Lubeck
DE
|
Family ID: |
34964065 |
Appl. No.: |
10/599942 |
Filed: |
April 15, 2005 |
PCT Filed: |
April 15, 2005 |
PCT NO: |
PCT/EP05/03978 |
371 Date: |
July 30, 2007 |
Current U.S.
Class: |
514/1.1 ;
435/7.21; 530/350; 530/388.1 |
Current CPC
Class: |
C07K 16/28 20130101;
G01N 33/6893 20130101; C07K 14/705 20130101; G01N 2800/065
20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/12 ; 530/350;
530/388.1; 435/7.21 |
International
Class: |
A61K 38/00 20060101
A61K038/00; C07K 14/00 20060101 C07K014/00; C07K 16/18 20060101
C07K016/18; A61P 43/00 20060101 A61P043/00; G01N 33/53 20060101
G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 15, 2004 |
DE |
10 2004 018 418.6 |
Claims
1. A goblet cell antigen, characterized in that the antigen is
obtainable through expression by the cell line HT29-18N2 which has
differentiated to goblet cells in protein-free medium, and is
detectable with anti-goblet cell antibodies from colitis ulcerosa
patients.
2. The goblet cell antigen as claimed in claim 1, characterized in
that the apparent molecular weight, measured in non-reducing
SDS-PAGE, is higher than 185 kDa.
3. The goblet cell antigen as claimed in claims 1, characterized in
that the apparent molecular weight of the non-reduced antigen in
agarose gel electrophoresis is between 950 kDa and 2,000 kDa.
4. The goblet cell antigen as claimed in claim 1, characterized in
that it is detectable with at least one of the lectins PHA-L,
PHA-E, RCA, Con A, LCA, PSA and AAL.
5. The goblet cell antigen as claimed in claim 1, characterized in
that it is detectable with the lectins PHA-L, PHA-E, RCA, Con A,
LCA, PSA and AAL.
6. The goblet cell antigen as claimed in claim 1, characterized in
that it is not detectable with the lectins SNA, HHL, MAL I, SBA,
DBA, UEA I, SJA, PNA, WGA, GSL I, PTL I, WGA s+b.
7. The goblet cell antigen as claimed in claim 1, characterized in
that it comprises galactose, N-acetyl-galactosamine and/or
fucose.
8. The goblet cell antigen as claimed in claim 1, characterized in
that it comprises N- and O-glycosidically bonded glycans.
9. The goblet cell antigen as claimed in claim 1, characterized in
that the glycans are partly or completely removed by chemical or
enzymatic methods.
10. The goblet cell antigen as claimed in claim 1, characterized in
that the reduced antigen comprises one or more glycosylated
proteins of the molecular weight 56, 66 or 80 kDa.
11. The goblet cell antigen as claimed in claim 1, characterized in
that the antigen is not detectable with antibodies from Crohn's
disease patients.
12. The goblet cell antigen as claimed in claim 1, characterized in
that the antigen is expressed by a eukaryotic cell line.
13. The goblet cell antigen as claimed in claim 1, characterized in
that the antigen is expressed by a human cell line.
14. The goblet cell antigen as claimed in claim 1, characterized in
that the antigen is expressed by a human colon carcinoma cell
line.
15. The goblet cell antigen as claimed in claim 1, characterized in
that the antigen is expressed by a cell line which is derived from
the cell line HT29.
16. The goblet cell antigen as claimed in claim 1, characterized in
that the antigen is expressed by the cell line HT29-18N2 which has
differentiated to goblet cells.
17. The goblet cell antigen as claimed in claim 1, characterized in
that the antigen is purified.
18. The goblet cell antigen as claimed in claim 1, characterized in
that the antigen is purified from the cell culture supernatant of
the cultured cell line.
19. The goblet cell antigen as claimed in claim 1, characterized in
that the antigen is purified from the cultured cell line.
20. A monoclonal antibody, characterized in that it recognizes the
goblet cell antigen as claimed in claim 1.
21. A kit for diagnosis of inflammatory bowel diseases,
characterized in that it comprises the goblet cell antigen as
claimed in claim 1 and optionally the monoclonal antibody as
claimed in claim 20.
22. A method for the detection of antibodies against goblet cell
antigen, in which a biological sample is contacted with the goblet
cell antigen as claimed in claim 1 and binding of antibody to the
antigen is detected.
23. The method as claimed in claim 22, characterized in that the
binding of the antibodies to the antigen is detected with an ELISA,
blot, western blot or dot blot.
24. A method for the detection of antibodies against goblet cell
antigen, in which a sample is contacted with a cell line which is
derived from the HT29 cell line and has differentiated to goblet
cells in protein-free medium, and the binding of antibody to the
antigen is detected as claimed in claim 1.
25. The method as claimed in claim 24, characterized in that the
cell line which has differentiated to goblet cells in protein-free
medium is the cell line HT29-18N2.
26. The method as claimed in claim 24, characterized in that the
binding of the antibodies to the antigen is detected with indirect
or direct immunofluorescence.
27. A method for diagnosis of inflammatory bowel diseases, in which
samples from patients are subjected to a method as claimed in claim
22, a colitis ulcerosa being diagnosed by detection of antibodies
against goblet cell antigen.
28. The use of goblet cell antigen as claimed in claim 1 for the
preparation of a pharmaceutical composition for treatment of
colitis ulcerosa.
29. The use of goblet cell antigen as claimed in claim 1 for ex
vivo removal of anti-goblet cell antibodies from the blood of
colitis ulcerosa patients.
30. The use as claimed in claim 28, characterized in that the
antigen is coupled to an inert matrix.
31. The use as claimed in claim 30, characterized in that the inert
matrix is silica gel, alginate, cellulose, pectin or carrageen.
32. The use as claimed in claim 28, characterized in that the
antigen is coupled to a cytotoxin.
33. The use as claimed in claim 32, characterized in that the
cytotoxin is specific for B lymphocytes.
34. A pharmaceutical composition for treatment of colitis ulcerosa,
characterized in that it comprises goblet cell antigen as claimed
in claim 1.
Description
[0001] The present invention relates to goblet cell antigen, a
method for detection of antibodies against goblet cell antigen
which is suitable for diagnosis of inflammatory bowel diseases, in
particular colitis ulcerosa, and a kit for diagnosis of
inflammatory bowel diseases, as well as monoclonal antibodies
against goblet cell antigen.
[0002] The significance of inflammatory bowel diseases has
increased in recent years due to an increasing incidence above all
in western industrialized countries (Jenss et al., 1996, Morbus
Crohn und Colitis ulcerosa. Informationen und Ratschlage, Serie
Gesundheit [Crohn's disease and colitis ulcerosa. Information and
advice, Health series] Piper/C & H.).
[0003] Colitis ulcerosa and Crohn's disease are chronic
inflammatory intestinal diseases (CIID) which are characterized by
inflammations which can affect various portions of the
gastrointestinal tract to different degrees. In the case of colitis
ulcerosa, in contrast to Crohn's disease, the inflammation reaction
is limited to the large intestine and is therefore an important
differentiating feature.
[0004] Both diseases are distinguished by an intermittent course,
in which active phases of the disease with sometimes severe
symptoms alternate with virtually symptom-free periods of time,
which can last months or even years. Colitis ulcerosa and Crohn's
disease are characterized by similar clinical symptoms.
Nevertheless, the therapy and the medicaments necessary for
alleviation differ for the two diseases. Differential diagnosis
therefore acquires a particular importance.
[0005] Serological analysis gives first indications of the
existence and severity of an inflammation. In this context, as a
rule an increase in the typical inflammation parameters, such as
blood sedimentation rate, C-reactive protein and leukocyte count,
is detected.
[0006] The diagnosis of an inflammatory bowel disease can be made
relatively quickly by the clinical symptoms (diarrhea and abdominal
pain). The demarcation of CIID such as colitis ulcerosa and Crohn's
disease from other diseases with similar clinical symptoms is
important here. An infection with typical diarrhea pathogens
(pathogenic Escherichia coli, Salmonellae etc.) can be ruled out by
a bacteriological analysis of the stool.
[0007] Celiac disease or sprue is also a chronic diarrhea disease.
However, this is caused by an intolerance of the small intestine
towards the cereal protein gluten in combination with the enzyme
transglutaminase and is characterized by atrophy of the mucosa of
the small intestine. Since celiac disease sometimes also causes
symptoms of an inflammatory bowel disease but cannot be treated
with medicaments, an accurate diagnosis is very important. In cases
of celiac disease, gluten acts together with transglutaminase as an
allergen which triggers an antigen-antibody reaction which leads to
destruction of the mucous membrane of the small intestine. With the
aid of a biopsy from the small intestine and a serological analysis
for autoantibodies against endomysium and gliadin by means of
indirect immunofluorescence, a reliable demarcation is
possible.
[0008] Sonography of the abdominal cavity is a relatively simple
diagnostic measure. Thickenings of the intestinal wall and
complications such as abscesses and fistulae (above all with
Crohn's disease) can be detected by this method. However,
sonography contributes to the diagnosis to only a small extent.
Radiological investigation with contrast media is much more
conclusive, but has been pushed into the background due to exposure
to radiation. Further imaging methods which can be employed are
computerized tomography (CT) and magnetic resonance tomography
(MRT), which likewise make it possible to show changes in the
region of the intestine.
[0009] The method of choice for differential diagnosis of colitis
ulcerosa or Crohn's disease is still coloscopy, including a biopsy.
In cases of colitis ulcerosa, a superficial inflammation is
present, which is characterized by a uniform spread and an aboral
increase and the formation of crypt abscesses. For Crohn's disease,
a transmural inflammation reaction with which segmental,
sharp-edged foci and granulomas occur is typical. An unambiguous
diagnosis can therefore be made in most cases via the biopsy. Since
patients with colitis ulcerosa are at increased risk of cancer of
the large intestine, an annual examination of the large intestine
by means of coloscopy is advised, in order to be able to resort to
suitable therapeutic measures as quickly as possible if a carcinoma
forms.
[0010] A further branch of diagnostics which is only rarely
utilized by gastroenterologists is serological analysis of
autoantibodies with the aid of an indirect immunofluorescence test
(IIFT). Here, use is made of the fact that patients with Crohn's
disease or colitis ulcerosa develop specific antibodies.
[0011] In the case of Crohn's disease, antibodies against the
exocrine pancreas can be detected with a prevalence of 39% with
IIFT; these do not occur in patients with colitis ulcerosa. Titers
above 1:10 are pathognomic for Crohn's disease. Pancreas tissue
from suitable primates serves as the substrate in this context, and
in the positive case a "net-like granular, sometimes also drop-like
fluorescence" is to be detected (Stocker et al., 1987, Scand J
Gastroenterol, 22: 41-52).
[0012] In one study, 76% of patients with colitis ulcerosa and 7%
of patients with Crohn's disease had antibodies against an unknown
antigen of the granulocytes. Swabs with human ethanol-fixed
granulocytes show, with a positive result in the indirect
immunofluorescence, a "smooth, sometimes also finely granular,
perinuclear fluorescence of the cytoplasm (pANCA)" (Stocker et al.,
1987, ibid).
[0013] In 70% of patients with Crohn's disease and 8% of specimens
from healthy blood donors, antibodies against Saccharomyces
cerevisiae can be found in indirect immunofluorescence. The
occurrence of these antibodies is independent of the development of
the anti-pancreas antibodies. 80% of Crohn's disease patients can
be detected by a combination of the two tests.
[0014] In 1959, autoantibodies against intestinal goblet cells were
discovered in sera of patients with colitis ulcerosa (Broberger et
al., 1959, J Exp Med, 110: 657-674), which could be responsible for
the characteristic decrease in these cells in the course of the
disease (Hayashi et al., 2001, Digestion 63: 28-31).
[0015] In one study, it was possible to specifically detect
anti-goblet cell antibodies in 28% of patients with colitis
ulcerosa (Stocker et al., 1987, ibid). The significance of these
autoantibodies for the pathogenesis is still unclear, as is the
structure of the antigen. A clarification of the antigen structure
could not only provide insights into the mechanism of the disease,
it could also be the basis for the development of targeted therapy
possibilities.
[0016] Human fetal intestinal tissue is ideal as a substrate for
diagnostic investigation for autoantibodies against intestinal
goblet cells by means of IIFT. Since the use is disputed from the
ethical point of view and rat tissue leads to misleading results
(Stocker et al., 1984, Deutsche Medizinische Wochenschrift, 51/52:
1963-1969), however, as a rule intestinal tissue from suitable
primates is employed in practice. In the positive case, a
cloud-like, blurred fluorescence can be seen in a plane above the
goblet cells (Stocker et al., 1987, ibid). In addition to ethical
considerations and the scarce availability of such substrates,
there are also difficulties with reliability, as a result of which
a very expensive and comprehensive quality control becomes
necessary, however, a 100% reproducible quality cannot be ensured
by these means, since it is a biological tissue which cannot be
influenced. Frequent non-specific reactions render evaluation of
the results difficult, so that these diagnostics can be carried out
by only a few experts.
[0017] On the basis of the difficulties described, differing
results have been achieved and published in the past. Investigation
of these antibodies is therefore scarcely used for diagnosis of
colitis ulcerosa, although the predictive value of a positive
result is virtually 100%, and a colitis ulcerosa can be
unambiguously detected with a positive test for anti-goblet cell
antibodies. By a combination of the evidence from anti-goblet cell
antibodies and pANCA, in 83% of the patients affected, colitis
ulcerosa could actually be diagnosed serologically.
[0018] The development of a reliable test system, which is easy to
evaluate and is based on a more easily accessible and more readily
standardizable source of the target structure for anti-goblet cell
antibodies, is therefore necessary in order to render a diagnosis
and subsequent specific therapy of colitis ulcerosa based on
investigation of these antibodies possible.
[0019] This problem is solved by the subject matter of claims 1 to
34, and in particular by the use of a goblet cell antigen which is
obtainable by culture of HT29-18N2 cells under certain culture
conditions and which reacts specifically with anti-goblet cell
antibodies from colitis ulcerosa patients.
[0020] Attempts have already been made earlier to develop cell
culture systems which allow a specific detection of antibodies in
cases of colitis ulcerosa or Crohn's disease. Lee et al. (1999, Gut
44: 196-202) investigated the three colon carcinoma cell lines
Caco-2, HT29 and LS-180 for their suitability as a source of the
target structure for anti-goblet cell antibodies for a specific
test system for detection of such antibodies. However, the results
obtained with this system were not specific for colitis ulcerosa
patients, but the cell lines investigated also reacted with
antibodies from Crohn's disease patients.
[0021] Furthermore, it was shown for the cell line HT29 that, by
exchange of glucose for galactose in the culture medium, it
apparently irreversibly differentiates into various intestinal
epithelial cells (Huet et al., 1987, JCB 105: 345-357). It was
shown for a subclone, HT29-18N2, that in addition to the morphology
of goblet cells, it also has some of their other properties, as,
e.g., the capability to be stimulated by carbachol, which induces
secretion of mucus. However, in other properties, the cell line
HT29-18N2 is said to differ from goblet cells, for example in its
growth properties or in the formation of intraepithelial lumina
(Phillips et al., 1988, Gastroenterology 94: 1390-1403).
[0022] Hibi et al. (1994, Gut 35: 224-230) attempted to employ the
cell line HT29-18N2 for diagnosis of inflammatory bowel diseases,
but did not arrive at specific results, since sera both of patients
with colitis ulcerosa (29-38%, depending on the method) and of
those with Crohn's disease (33%) reacted with the cells used. The
antigen recognized by the antibodies is said to have a molecular
weight of >200 kDa, to comprise an individual polypeptide chain
and to be recognized by the antibodies both in the native and in
the reduced form. Further identification of this antigen was not
undertaken.
[0023] It has furthermore been found that during culture in
glucose-containing (3.0 g/l), protein-free medium (e.g. Protein
Free Hybridoma Medium II, PFHM II, Invitrogen), the cell line
HT29-18N2 predominantly grows as goblet cells filled with secretory
granula, which, in contrast to the cell line HT29, form individual
layers (Phillips et al., 1995, In Vitro Cell Dev Biol 31: 421-423).
These cells moreover form a number of glycoproteins, so-called
mucins, which are in some cases secreted.
[0024] Surprisingly, it has now been found that during culture of
HT29-18N2 cells in the absence of protein, an antigen which reacts
with anti-goblet cell antibodies from colitis ulcerosa patients is
expressed. This antigen is called goblet cell antigen in the
following.
[0025] The present invention for the first time renders targeted
preparation and purification of goblet cell antigen possible.
[0026] Therefore, in the context of this invention, goblet cell
antigen is provided, which is obtainable by expression by the cell
line HT29-18N2 which has differentiated to goblet cells in
protein-free medium, as disclosed, e.g., in WO8802774 or WO9808934,
or in PFHM II medium (Protein Free Hybridoma Medium, Invitrogen),
and which is detectable with anti-goblet cell antibodies from
colitis ulcerosa patients.
[0027] The goblet cell antigen can be expressed, e.g., by the cell
line HT29-18N2 which has differentiated to goblet cells. This cell
line has already been used many times in research and is therefore
readily obtainable, e.g. from T. Phillips (University of Missouri,
Columbia, USA), Euroimmun (Euroimmun A G, Seekamp 31, 23560 Lubeck,
Germany), B. L. Rodriguez (Departamento de Genetica, CNIC, Havanna,
Cuba), R. A. Finkelstein (University of Missouri, Columbia, USA),
J. A. Benitez (Morehouse School of Medicine, Atlanta, USA), A. Frey
(Forschungszentrum Borstel, 23845 Borstel, Germany), D. Louvard
(Institut Pasteur, 75724 Paris Cedex 15, France), R. J. Nijman
(Erasmus Medical Center, 3000 D R Rotterdam, The Netherlands), K.
Kobayashi (Veterans Affairs Medical Center, Denver, USA) or T. Hibi
(School of Medicine, Keio University, Japan).
[0028] A culture of HT29-18N2 cells in PFHM II (Protein Free
Hybridoma Medium II, Invitrogen) is particularly suitable for
differentiation to goblet cells which express the goblet cell
antigen. The cells can also be seeded in protein-containing medium
(e.g. DMEM (Dulbecco's Modified Eagle's Medium), 10% (v/v) fetal
calf serum (FCS), and the change to PFHM II leads to a change in
morphology to that of goblet cells. The cells are cultured for at
least 2 days, particularly preferably at least 4, 7, 10 or 14 days
in the absence of protein.
[0029] The cell line HT29-18N2 can be obtained by differentiation
of the cell line HT29 in the absence of glucose by the method of
Huet et al. (Huet et al., 1987, ibid).
[0030] The HT-29 cells are a cell line of a human colon
adenocarcinoma, which can be obtained e.g. from DSMZ (Deutsche
Sammlung fur Mikroorganismen und Zellkulturen GmbH, deposition
number ACC299). The adherent, epitheloid cells grow in multiple
layers and in large colonies. The development of these
non-differentiated precursors of intestinal wall cells can be
influenced by the nature of the culture. If glucose in the medium
is replaced by galactose, the cells differentiate to enterocytes
(approx. 90%) and goblet cells (approx. 10%) in a period of time of
from approx. 1 to 2 weeks (Huet et al., 1987, ibid). Further
subclones with the properties of the HT29-18N2 clone, that is, also
expression of the goblet cell antigen, can be obtained from the
parent cell line by the method demonstrated in the publication by
Huet et al. Goblet cell antigen can also be obtained from such cell
lines under suitable culture conditions.
[0031] It has furthermore been found that daughter cell lines
(HT29-MTX, HT29-FU, HT29-5M21, HT29-5F7) with some of the
properties of goblet cells, as, e.g., the production of mucins
typical of goblet cells, can be obtained from the cell line HT29 by
culture in the presence of methotrexate and/or 5-fluorouracil
(Lesuffleur et al., 1991, Int J Cancer, 49: 731-737, Leteurtre,
2004, Biol Cell, 96, 145-151). Goblet cell antigen can also be
obtained from these or analogously obtained cell lines using
suitable culture conditions.
[0032] The goblet cell antigen is particularly suitable for
diagnosis of colitis ulcerosa, since it is recognized specifically
by antibodies from the serum of approx. 28% of colitis ulcerosa
patients. Since not all colitis ulcerosa patients have antibodies
against goblet cell antigen (Stocker et al., 1987, ibid), for
identification of the goblet cell antigen according to the
invention it is necessary to use either sera from colitis ulcerosa
patients which have tested positively for reactivity with goblet
cell antigen, e.g., on primate intestine (see e.g. Example 1) or
antibodies from a sufficient number of colitis ulcerosa patients,
if appropriate in purified form. Anti-goblet cell antibodies from
colitis ulcerosa patients correspond to anti-goblet cell-positive
serum from colitis ulcerosa patients. Where appropriate, antibodies
from serum which has tested positive can be purified before use as
a positive control, e.g. via a protein A or G column.
[0033] The goblet cell antigen according to the invention is
preferably not detectable with antibodies from Crohn's disease
patients. It is furthermore preferably also not detectable with
antibodies from patients with other autoimmune diseases (apart from
colitis ulcerosa). In the studies carried out to date, no
cross-reactivity of antibodies from patients with Crohn's disease
or other autoimmune diseases with the goblet cell antigen was
found. The presence of antibodies against the goblet cell antigen
is thus specific to colitis ulcerosa.
[0034] It was possible to demonstrate that both the cells and the
culture supernatant contain the goblet cell antigen. It is
therefore partly located extracellularly. These results explain the
localization of the immunofluorescence, e.g. in primate intestine,
with which a cloud-like, blurred fluorescence can be seen in a
plane above the goblet cells (Stocker et al., 1987, ibid). The
location within the cells can be illustrated only poorly by this
method, since the associated fluorescence is masked by the
overlying fluorescence. Nevertheless, the localization can be
demonstrated with a confocal laser scanning microscope which is
capable of fading out the interfering fluorescence.
[0035] In a particularly preferred embodiment, the goblet cell
antigen is expressed by the cell line HT29-18N2 which has
differentiated to goblet cells in PFHM II. Preferably, the antigen
is purified, in particular isolated, and is purified from the cell
culture supernatant of the cell line HT29-18N2 which has
differentiated to goblet cells in PFHM II.
[0036] A particularly simple, but adequate method for purifying the
antigen is separation of the culture supernatant from the cells.
The purification can be improved by removal of small proteins and
low molecular weight constituents of the medium, such as, for
example, biotin, e.g. by ultrafiltration or gel filtration.
Preferably, the antigen is separated from these constituents by
ultrafiltration with a filter having an exclusion size of at least
5 kDa, in particular of at least 100 kDa, and remains in the
retentate.
[0037] Of course, further purification of the antigen can be
carried out, e.g., by chromatography. Preferably, the antigen is
separated from other substances by ion exchange chromatography,
particularly preferably by an anion exchange chromatography at a pH
of .gtoreq.5.0, preferably at a pH of .gtoreq.7.5, by elution with
at least 150 mM NaCl, preferably by elution with at least 200 mM
NaCl.
[0038] Goblet cell antigen purified via anion exchange
chromatography has been characterized by various methods. In an
investigation by SDS-PAGE and western blotting with anti-goblet
cell-positive serum from colitis ulcerosa patients, only an
individual band, which lies above the top band of the marker (185
kDa), manifests itself under non-reducing conditions in the goblet
cell antigen-positive fractions. The goblet cell antigen thus
preferably has, measured in non-reducing SDS-PAGE, an apparent
molecular weight which is greater than 185 kDa.
[0039] Under reducing conditions, on the other hand, the sera do
not react with the goblet cell antigen. The epitopes recognized in
the target antigen are therefore probably mainly conformational in
nature, since they seem to be at least partly dependent on the
three-dimensional structure of the antigen and/or contain disulfide
bridges.
[0040] The results furthermore show that the goblet cell antigen
has a very high molecular weight. In order to be able to show
proteins of this size in differentiated form, agarose gel
electrophoreses with a separating range of from approx. 100 to
.gtoreq.2,000 kDa had to be carried out instead of the
polyacrylamide gel electrophoreses conventional in protein
biochemistry.
[0041] Under these conditions, the mobility of the goblet cell
antigen is below that of human IgM (non reduced, molecular weight
approx. 950 kDa, L. Stryer, Biochemie [Biochemistry],
Spektrum-Verlag, 4th edition 1995, p. 395). However, the mobility
is above that of the non-reduced mucin MUC5AC. The apparent
molecular weight of the non-reduced goblet cell antigen in agarose
gel electrophoresis is thus between 950 kDa and that of the
non-reduced MUC5AC (>2,000 kDa, see J. Bara et al., Biochem J,
15, 185-193). The apparent molecular weight can therefore be
estimated at greater than 1,000 kDa.
[0042] In the preparation, the goblet cell antigen appears to make
up the majority of the protein present. Two further protein bands
are detectable, one of the contaminating proteins being the mucin
MUC5AC, which is recognized by a monoclonal anti-MUC5AC antibody.
The other contaminating protein appears to have an apparent
molecular weight corresponding to that of non-reduced IgM (approx.
950 kDa) in the non-reduced state, since, in agarose gel, it has a
mobility similar to that of non-reduced human IgM.
[0043] In a western blot using lectins, a protein of the same
mobility was bound by various lectins. Here also, the conclusion
that this is the goblet cell antigen is suggested. The goblet cell
antigen is therefore preferably detectable with at least one or all
of the lectins PHA-L (Phaseolus vulgaris leukoagglutinin), PHA-E
(Phaseolus vulgaris erythroagglutinin), RCA (Ricinus communis
agglutinin), Con A (concanavalin A), LCA (Lens culinaris
agglutinin), PSA (Pisum sativum agglutinin) and AAL (Aleuria
aurantia lectin). Under the conditions used, however, the goblet
cell antigen was not detectable with the lectins SNA (Sambucus
nigra lectin), HHL (Hippeastrum hybrid lectin), MAL I (Maackia
amurensis lectin I), SBA (soybean agglutinin), DBA (Dolichus
biflorus agglutinin), UEA I (Ulex europaeus agglutinin), SJA
(Sophora japonica agglutinin), PNA (peanut agglutinin), WGA (wheat
germ agglutinin), GSL I (Griffonia simplicifolia lectin I), PTL I
(Psophocarpus tetragonolobus lectin I), WGA s+b (wheat germ
agglutinin, succinylated and biotinylated). On the other hand, the
contaminating proteins from the anion exchange chromatography all
appear not to react with the lectins investigated.
[0044] The specificity of the lectins which bind to the goblet cell
antigen shows that the antigen comprises galactose, glucose,
mannose, N-acetyl-galactosamine and/or fucose (see Table 1, below).
However, the present invention also provides non-glycosylated or
only partly glycosylated goblet cell antigen which is bound by
anti-goblet cell-positive serum.
[0045] In contrast to the anti-goblet cell-positive serum, after
reduction of the goblet cell antigen the lectins also bind to the
glycans which are uninfluenced by the reduction. The mobility of
the constituents detected is increased significantly compared with
the non-reduced goblet cell antigen, and the band with the previous
mobility disappears completely on reduction.
[0046] It can therefore be assumed that the goblet cell antigen
comprises several peptide chains which are bonded to one another
with disulfide bridges in the native state. In a reducing SDS-PAGE,
it was therefore possible to carry out a determination of the
apparent molecular weight of glycosylated proteins which appear to
be constituents of the goblet cell antigen. This indicates that the
goblet cell antigen comprises one or more glycosylated proteins of
molecular weight 52-54, 56, 66 and/or 80 kDa.
[0047] By preparation of the non-reduced total protein from an
agarose gel at the position of the band which becomes visible after
detection with anti-goblet cell-positive serum, subsequent
reduction followed by SDS-PAGE and western blotting with the
lectins which bind to the goblet cell antigen, it becomes clear
that only proteins having apparent molecular weights of 56, 66,
and/or 80 kDa are to be found at this position. In contrast, the
proteins in the range corresponding to 52-54 kDa are not present.
It can be concluded from this that the goblet cell antigen
comprises one or more proteins of the apparent molecular weights
56, 66, and/or 80 kDa.
[0048] After cleavage with N-glycosidase F, the apparent molecular
weight of these constituents was reduced to a small degree, but the
reactivity with the corresponding lectins was retained. From this
finding it can be deduced that the goblet cell antigen comprises N-
and O-glycosidically bonded glycans. Furthermore, the carbohydrates
detectable via the lectins, which are partly terminal
carbohydrates, can in some cases be removed by specific
glycosidases and the underlying carbohydrates are exposed. In a
preferred form, the glycans of the goblet cell antigen are
therefore partly or completely removed by chemical or enzymatic
methods.
[0049] Further purification of the goblet cell antigen, e.g. from
the positive fractions of the anion exchange chromatography, is
possible by standard methods, for example size fractionation by
means of gel filtration or HPLC.
[0050] However, purification with the aid of affinity
chromatography with antisera from the blood from colitis ulcerosa
patients or with the aid of the lectins which bind to the goblet
cell antigen is particularly preferred. For the person skilled in
the art, further methods result for the purification and isolation
of the goblet cell antigen from the culture supernatant or from
cells, such as, for example, filtration, chromatography,
electrophoresis and centrifugation methods or combinations of
these. Such methods of purification of proteins are described e.g.
in Lottspeich and Zorbas (Bioanalytik [Bioanalysis], 1998, Spektrum
akademischer Verlag, Heidelberg, Berlin) or Sambrook et al.
(Molecular cloning: a laboratory manual, 2000, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.). The present invention
therefore also provides goblet cell antigen which is purified or
isolated by such methods and is bound by anti-goblet cell-positive
serum.
[0051] With the aid of the purified antigen, the DNA sequence which
codes for it can be determined by known methods. Thus, e.g.,
sequencing of the purified protein fragments of the goblet cell
antigen allows synthesis of degenerated primers with which the cDNA
which codes for goblet cell antigen can be identified in a cDNA
library, e.g., from the HT29-18N2 cells cultured in PFHM II.
Screening of a cDNA expression library with antibodies generated
against goblet cell antigen or with antibodies from colitis
ulcerosa patients or the search in suitable databanks is also
possible.
[0052] Cloning of the cDNA into an expression vector allows
recombinant expression of the antigen. Expression of the protein is
possible in various systems, e.g. bacterial systems, such as
Escherichia coli or Bacillus subtilis, in yeasts, such as
Saccharomyces cerevisiae or Pichia pastoris, or other eukaryotic
cells, such as 293, CHO, COS or insect cells.
[0053] Expression in eukaryotic cells, preferably in a eukaryotic
cell line, is preferred, since, here, a glycosylation of the
protein and necessary further processing steps can take place.
Various expression vectors are known for the particular
systems.
[0054] In a preferred embodiment, the goblet cell antigen is
expressed as a fusion protein, e.g. as Ig fusion protein or using a
tag for protein purification, e.g. a polyhistidine tag (His tag).
Accordingly, in a preferred embodiment, a recombinant antigen is
used. The mentioned methods are described, e.g., in Lottspeich and
Zorbas (Bioanalytik [Bioanalysis], 1998, Spektrum akademischer
Verlag, Heidelberg, Berlin) or Sambrook et al. (Molecular cloning:
a laboratory manual, 2000, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.).
[0055] In a particularly preferred embodiment, only the parts of
the antigen to which the antibodies from colitis ulcerosa patients
bind are expressed, in order to avoid, e.g., non-specific binding
of antibodies from patients with other autoimmune diseases or to
optimize the expression of the antigen. Such parts can comprise
either individual reactive regions of the antigen, or fusion
proteins from several reactive regions of the antigen or fusion
proteins from one or more reactive regions of the antigen, and
sequences which do not belong to the goblet cell antigen.
[0056] The present invention also provides a monoclonal antibody
which recognizes goblet cell antigen. A preferred method for
obtaining such an antibody is immunization of a mouse with purified
goblet cell antigen. After approx. 2 weeks, the spleen is removed
and the spleen cells are fused with myeloma cells in the presence
of polyethylene glycol (PEG). The myeloma cells lack the enzyme
HPRT, so they cannot survive in HAT selection medium.
[0057] Only myeloma cells fused with spleen cells survive culture
in the selection medium. The hybridoma cells formed are cloned and
tested for the production of antibodies with the aid of the method
according to the invention. The method for the preparation of
monoclonal antibodies is described in detail, e.g., in Sambrook et
al. (Molecular cloning: a laboratory manual, 2000, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
[0058] A particularly preferred method comprises humanization of
the monoclonal antibody obtained in this way. This is effected by
isolating the associated cDNA from the hybridoma cells and
exchanging the sequence which codes for the F.sub.c part of the
monoclonal antibody by a corresponding human sequence. The hybrid
sequence formed in this way can be transferred into a suitable
expression vector and expressed in a suitable expression system and
purified from this by known methods. In a preferred embodiment, a
recombinant antibody is accordingly used. The methods mentioned are
described, e.g., in Lottspeich and Zorbas (Bioanalytik
[Bioanalysis], 1998, Spektrum akademischer Verlag, Heidelberg,
Berlin) or Sambrook et al. (Molecular cloning: a laboratory manual,
2000, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.).
[0059] A further preferred method for obtaining a monoclonal
antibody according to the invention is isolation of specific human
B lymphocytes from inflamed intestinal tissue from colitis ulcerosa
patients with anti-goblet cell-positive serum. Such lymphocytes can
be obtained by excision of the intestinal region affected and
culture of the cells present, in particular the B lymphocytes, in a
suitable medium. Further isolation of specific B lymphocytes can
take place by sorting the cells obtained in a fluorescence-assisted
cell sorter (FACS) with the aid of a B lymphocyte-specific antibody
labeled with a fluorophore, e.g. anti-CD20, for identification of B
lymphocytes, and/or of the purified goblet cell antigen labeled
with a second fluorophore, for identification of specific B
lymphocytes. These can be converted and selected, e.g., in a manner
analogous to the method described above, into hybridoma cells.
[0060] An alternative possibility is the establishment of a phage
display library based on the cDNAs of the antibodies from the cells
obtained in this way. After screening for binding to the goblet
cell antigen purified on the basis of the method according to the
invention, a specific monoclonal antibody can be obtained by
isolating the cDNA which codes for the specific immunoglobulin,
conversion of the cDNA into a suitable expression vector,
expression in a suitable expression system and purification of the
antibody by known methods. In a preferred embodiment, a human
recombinant antibody is accordingly used. The methods mentioned are
described e.g. in Eggena et al., 1996, J Immunol, 156:
4005-4001.
[0061] A further preferred method for obtaining a monoclonal
antibody according to the invention is screening of a phage display
library based on randomized cDNAs of human antibodies, e.g. the
HuCAL Gold Library from MorphoSys, for binding to the purified
goblet cell antigen with one of the methods according to the
invention. By subsequent transfer of the associated cDNAs into a
suitable expression vector, expression in a suitable expression
system, purification of the antibody by known methods and screening
for desired properties, a Fab fragment specific for the goblet cell
antigen can be obtained first. Accordingly, in a preferred
embodiment, a human recombinant Fab fragment is used. This can be
modified by complementation with a cDNA which codes for the F.sub.c
part, to give a complete human immunoglobulin.
[0062] Complete immunoglobulins can also be obtained directly in an
analogous manner using other libraries. The methods mentioned are
described in the technical description of the HuCAL Gold Library of
MorphoSys.
[0063] Further methods for preparation of monoclonal antibodies are
evident for the person skilled in the art. The present invention
therefore also provides monoclonal antibodies which are prepared by
such methods and bind to goblet cell antigen which is bound by
anti-goblet cell-positive serum.
[0064] Furthermore, a kit for diagnosis of inflammatory bowel
diseases which comprises goblet cell antigen and, e.g.,
instructions for diagnosis of inflammatory bowel diseases is
provided in the context of the invention. Preferably, the kit
furthermore also comprises the monoclonal antibody, so that
positive sera from colitis ulcerosa patients do not have to be used
for carrying out the method according to the invention.
[0065] A method for detecting antibodies against goblet cell
antigen, in which a biological sample is contacted with an antigen
and binding of the antibody to the antigen is detected, is also
provided in the context of this invention, the antigen being
obtainable by expression by the cell line HT29-18N2 which has
differentiated to goblet cells in PFHM II and being detectable with
anti-goblet cell antibodies from colitis ulcerosa patients. The
goblet cell antigen described above is employed for the method.
[0066] The method is particularly suitable for diagnosis of colitis
ulcerosa, since the antigen is specifically recognized by
antibodies from the serum of approx. 28% of colitis ulcerosa
patients. The sample which is to be analyzed for antibodies against
goblet cell antigen is a biological sample, in general a blood or
serum sample.
[0067] Preferably, it is a sample from a patient with an
inflammatory bowel disease, in whom colitis ulcerosa could be
present. However, it can also be e.g. the supernatant of a
hybridoma or a phage display library.
[0068] In the method according to the invention, various methods
can be employed for detecting the binding of the antibodies to the
antigen. This is preferably carried out with an ELISA or blot, e.g.
western blot or dot blot.
[0069] Moreover, rapid tests for qualitative detection of
antibodies for reading a color reaction on a test strip can also be
used. Such tests are in general based on a sandwich solid phase
immunoassay, in which goblet cell antigen is coupled to a test
field or in a line on the test strip. The test strip is brought
into contact, e.g., with a blood sample or diluted patient serum,
which possibly contains anti-goblet cell antigen antibodies.
Secondary anti-human immunoglobulin antibodies of suitable
specificity, e.g. conjugated with gold conjugate or with an enzyme,
such as alkaline phosphatase or horseradish peroxidase, collect in
the test field only if the sample contained antibodies. If
appropriate, the result must also be rendered visible with a
chromogenic substrate.
[0070] The present invention furthermore provides a method for
detection of antibodies against goblet cell antigen, in which a
sample is contacted with a cell line which is derived from the HT29
cell line and has differentiated to goblet cells in protein-free
medium, and the binding of antibodies to the antigen is detected.
The culture conditions which are preferably used for this have
already been described in detail above. The goblet cell antigen
according to the invention expressed by the cells under these
culture conditions specifically reacts with anti-goblet cell
antibodies from colitis ulcerosa patients.
[0071] It is thus not detectable with antibodies from Crohn's
disease patients, and preferably also not with antibodies from
patients with other autoimmune diseases. Preferably, the cell line
which has differentiated to goblet cells in protein-free medium is
the cell line HT29-18N2.
[0072] Carrying out the method according to the invention with the
cells described allows detection of the binding of the antibodies
to the antigen with indirect or direct immunofluorescence. This
method is currently the standard method for detecting goblet cell
antigen (Stocker et al., 1984, 1987, ibid) using intestinal tissue,
so that a comparison of the methods is particularly easily
possible. This comparison shows that with anti-goblet cell-positive
sera, the cells show a cloud-like, blurred fluorescence in a plane
above the cells which is very similar to the reaction of the same
sera on primate and human fetal intestine.
[0073] By comparison, blood donor sera showed no specific
reactivity. A comparison of the sensitivity of the substrates in
indirect immunofluorescence shows that the HT29-18N2 cell line is
more suitable than primate intestine as a substrate for diagnostics
for detecting anti-goblet cell antibodies. Non-specific reactions
are absent or are negligibly low, so, at any time, results which
are comparable and can be evaluated unambiguously can be achieved
in the diagnosis of colitis ulcerosa with the indirect
immunofluorescence test with HT29-18N2 cells as the substrate.
[0074] The prevalence of antibodies against goblet cell antigen
determined hitherto with the method according to the invention is
approx. 28%. This is in agreement with the values already
determined by Stocker et al. (1987, ibid). Diseases with proven
autoimmune pathogenesis also show a prevalence of the corresponding
autoantibodies of less than 90% in most cases.
[0075] The relatively low percentage of anti-goblet cell antibodies
with colitis ulcerosa could be caused by a neutralization of the
circulating antibodies by erythrocytes or other cells or
structures: It has, e.g., been found that patients with blood group
0 show a prevalence of only 19%, while in patients with blood group
AB it is 66%.
[0076] The predictive value of a detection of antibodies against
goblet cell antigen is 100%. A method for diagnosis of inflammatory
bowel diseases in which these antibodies are detected with the
method according to the invention is therefore provided in the
context of the invention, wherein a colitis ulcerosa is diagnosed
by detection of antibodies against goblet cell antigen.
[0077] Reliable test systems have already been established for
anti-pancreas, anti-Saccharomyces cerevisiae antibodies and pANCA,
and the present invention provides such a test system for
anti-goblet cell antigen. A combination of the four tests would in
many cases make endoscopy superfluous. In approx. 80. % of patients
with chronic inflammatory bowel diseases, a differential diagnosis
can thus already be made serologically by investigation of the
antibodies against goblet cell antigen, pancreas secretion,
Saccharomyces cerevisiae and pANCA.
[0078] The present invention also provides the use of goblet cell
antigen for the preparation of a pharmaceutical composition for
treatment of colitis ulcerosa. The present invention furthermore
provides a pharmaceutical composition, preferably for treatment of
colitis ulcerosa, which comprises goblet cell antigen, optionally
with a pharmaceutically suitable carrier agent and/or
auxiliaries.
[0079] In the treatment of colitis ulcerosa, for example, a body
fluid of the patient is brought into contact with the antigen.
[0080] In the context of this invention, body fluid is understood
as meaning, e.g., blood or intestinal content or the secretion of
the mucous membrane of the patient. Preferably, goblet cell antigen
is coupled to an inert matrix, e.g., silica gel or a carbohydrate
which is indigestible by humans, such as alginate, cellulose,
pectin, carrageen or the like. This conjugate is then brought into
contact with a body fluid of a patient with anti-goblet cell
antibodies in order to bind the anti-goblet cell antibodies
contained in this body fluid to the conjugate.
[0081] There are various possibilities for bringing the body fluid
of a patient into contact with such a matrix. One possibility is
haemodialysis to remove the antibodies. In this context, blood of a
colitis ulcerosa patient is taken from the body and brought into
contact with the conjugated matrix. The blood is then separated
from the conjugate and returned, so that the anti-goblet cell
antibodies are removed from the body fluid and can no longer
participate in the inflammatory process. Since most of the
anti-goblet cell antibodies are present as IgG, removal of the
specific antibodies of this isotype present in the blood
significantly reduces the total amount of antibodies. The present
invention therefore also provides the use of goblet cell antigen
for ex vivo removal of anti-goblet cell antibodies from the blood
of colitis ulcerosa patients.
[0082] In addition to such a contact ex vivo, an in vivo contact is
also possible. For example, an inert matrix with coupled goblet
cell antigen can be brought into the intestine through the stomach
in tablet form, that is to say in a formulation resistant to
gastric juice.
[0083] Coupling of a cytotoxin to the goblet cell antigen is
furthermore preferred. After administration to colitis ulcerosa
patients with anti-goblet cell antibodies, the conjugate binds to
the specific B lymphocytes and destroys these or induces apoptosis
in them. Preferably, the cytotoxin is specific for B
lymphocytes.
[0084] Alternatively, a pharmaceutical composition which comprises
the goblet cell antigen can also be used for inducing tolerance to
the goblet cell antigen. Preferably, for this, the goblet cell
antigen is formulated with antiinflammatory cytokines, such as e.g.
TGF-.beta. or IL-10, which can induce regulatory T cells (Chen et
al., 2003, J. Exp. Med., 198, 1875-86). Suitable adjuvants and
further methods for inducing tolerance are known in the prior art,
e.g. blocking co-stimulating signals or administration of the
antigen in the context of tolerogenic autologous dendritic cells
(Xiao et al., 2003, BioDrugs, 17, 103-11).
[0085] The following experiments show particular embodiments of the
invention. It was first attempted to employ human or primate
intestinal tissue for isolation of goblet cell antigen. As already
known in the literature, this tissue is suitable for detection of
anti-goblet cell antibodies by means of indirect
immunofluorescence. However, it was found that with none of the
experiments carried out (western blotting, neutralization tests,
lectin ELISA), specific reactions of one or more fractions of an
intestine preparation with the anti-goblet cell-positive sera can
be detected. Under the given conditions, intestinal tissue
therefore does not appear to be suitable for purification of goblet
cell antigen and preparation of standardized test systems based on
an ELISA or blotting technique. Analogous results were achieved
with the cell line HT29 cultured under standard conditions (see
information on the cell line of the Deutsche Sammlung fur
Mikroorganismen [German Collection of Microorganisms]).
[0086] Thus, the cell line HT29-18N2 was subsequently investigated,
which was found, surprisingly, to express goblet cell antigen under
the culture conditions described above. Both indirect
immunofluorescence and ELISA and western blotting were possible
with this cell line, so it is suitable for detecting anti-goblet
cell antibodies. Furthermore, goblet cell antigen can be purified
with the aid of this cell line.
LEGEND TO THE FIGURES
[0087] FIG. 1: Indirect immunofluorescence with intestinal
tissue
[0088] A: Representation of autoantibodies from colitis ulcerosa
patients against intestinal goblet cells (anti-goblet cell
antibodies) by indirect immunofluorescence on human fetal intestine
(200-fold magnification).
[0089] B: Representation of autoantibodies against intestinal
goblet cells (FITC-labeled) and anti-MUC2 antibodies
(TRITC-labeled) by indirect immunofluorescence on prim. adult
intestine (200-fold magnification).
[0090] C, D: Representation of the anti-MUC2 antibody by indirect
immunofluorescence in prim. adult intestine (C: 200-fold
magnification, D: 400-fold magnification).
[0091] FIG. 2: Fractionation of intestine tissue
[0092] A: Silver gel of the fractions of a preparation from human
adult intestinal tissue. In each case 1 .mu.l of the stated
fractions was applied. The samples were obtained by centrifugation
at increasing revolutions per minute.
[0093] B, C: Western blot of the fractions of the intestinal
preparation, incubated with a mixture of anti-goblet cell-positive
sera (B) and blood donor sera (C) (1: homogenate, 2: supernatant 1,
3: sediment 1, 4: supernatant 2, 5: sediment 2, 6: supernatant 3,
7: sediment 3, 8: supernatant 4, 9: sediment 4, 10: supernatant 5,
11: sediment 5).
[0094] FIG. 3: Diagram of the structure of a lectin ELISA
[0095] The biotinylated lectins bind to a plate coated with
streptavidin and blocked with BSA. Antigens present in the sample
are bound via their carbohydrate content and a reaction with
primary antibodies from the serum and POD-labeled secondary
antibodies from the conjugate is specifically detected.
[0096] FIG. 4: Indirect immunofluorescence with HT29-18N2 cells
[0097] A, B: Representation of autoantibodies against goblet cells
(A), of a blood donor serum (B), of anti-MUC2 antibodies (C) and of
anti-MUC5AC antibodies (D) by indirect immunofluorescence on
HT29-18N2 cells after differentiation to goblet cells in PFHM II
medium (200-fold magnification).
[0098] FIG. 5: Analysis of HT29-18N2 cell culture supernatants
[0099] A: Representation of the reactivity of anti-goblet
cell-positive sera (serum 1 to 3) compared with blood donor sera
(BD 1 to 4) and the anti-MUC5AC antibody by dot blotting.
[0100] The samples were concentrated with a filter having an
exclusion size of 100 kDa and the medium was replaced by PBS. In
each case 2 .mu.l of sample were applied. The samples were cell
culture supernatants from various culture bottles (A, B and C)
obtained at different time points after the start of the
differentiation.
[0101] B: Representation of the reactivity of anti-goblet
cell-positive sera and blood donor sera (BD sera) compared with
chromatography fractions of an HT29-18N2 cell culture
supernatant.
[0102] In each case 5 .mu.l of the fraction were applied.
[0103] The right-hand strip shows the sequence of fractions applied
(A means application). The negative control was incubated with
buffer instead of a serum dilution.
[0104] FIG. 6: Detection of antibodies against goblet cell antigen
by ELISA
[0105] A: Reactivity of various starting substrates (cells and cell
culture supernatant) with anti-goblet cell antibodies from sera of
colitis ulcerosa patients (CU, dark) and blood donor sera (BD,
light) in ELISA.
[0106] B: Reactivity of the chromatography fractions of a cell
culture supernatant with anti-goblet cell-positive and blood donor
sera in ELISA.
[0107] FIG. 7: Characterization of goblet cell antigen in
SDS-PAGE/western blot
[0108] A: SDS-PAGE/western blot of the chromatography fractions
under reducing/non-reducing conditions
[0109] The same amounts of a sample of a chromatography fraction
positive for goblet cell antigen were used under reducing
(left-hand track each time) and non-reducing (right-hand track each
time) conditions for SDS-PAGE. The western blot was stained with
anti-MUC5AC antibody (middle strip) and anti-goblet cell-positive
serum (right-hand strip). MultiMark was used as the marker.
[0110] B: Agarose gel electrophoresis/western blot of the
chromatography fractions under reducing/non-reducing conditions
[0111] The same amounts of a sample of a chromatography fraction
positive for goblet cell antigen were used under reducing
(left-hand track each time) and non-reducing (right-hand track each
time) conditions for an agarose gel electrophoresis. The western
blot was stained with anti-goblet cell-positive sera (1st and 2nd
strip from the left), blood donor sera (3rd and 4th strip from the
left) and anti-MUC5AC antibody (right-hand strip).
[0112] C: FITC labeling of a goblet cell antigen-positive
chromatography fraction
[0113] The sample of the chromatography fraction was labeled with
FITC and analyzed under reducing (lane 2) and non-reducing (lane 3)
conditions. A non-labeled, non-reduced sample, detected with
anti-goblet cell antigen-positive serum (lane 4) or anti-MUC5AC
antibody (lane 5) and non-reduced human IgM (lane 1) serve as a
comparison.
[0114] FIG. 8: Characterization of the glycans of goblet cell
antigen by western blotting with lectins
[0115] A: Agarose gel electrophoresis/western blot of the
chromatography fraction under non-reducing conditions
[0116] Strip 1 (from left to right): anti-goblet cell-positive
serum; strip 2 and 3: blood donor sera; strip 4: SNA; strip 5:
PHA-L; strip 6: PHA-E; strip 7: HHL; strip 8: RCA; strip 9: MAL I;
strip 10: Con A; strip 11: SBA; strip 12: DBA; strip 13: UEA I;
strip 14: SJA; strip 15: PNA; strip 16: WGA; strip 17: LCA; strip
18: GSL I; strip 19: PSA; strip 20: PTL I; strip 21: AAL; strip 22:
WGA s+b.
[0117] B: Agarose gel electrophoresis/western blot/lectin blot of
the chromatography fraction under reducing conditions
[0118] Strip 1 (from left to right): SNA; strip 2: PHA-L; strip 3:
PHA-E; strip 4: HHL; strip 5: RCA; strip 6: MAL I; strip 7: Con A;
strip 8: SBA; strip 9: DBA; strip 10: UEA I; strip 11: SJA; strip
12: PNA; strip 13: WGA; strip 14: LCA; strip 15: GSL I; strip 16:
PSA; strip 17: PTL I; strip 18: AAL; strip 19: WGA s+b.
[0119] C: SDS-PAGE/western blot/lectin blot of the chromatography
fraction under reducing conditions
[0120] Strip 1 (from left to right): SNA; strip 2: PHA-L; strip 3:
PHA-E; strip 4: HHL; strip 5: RCA; strip 6: MAL I; strip 7: Con A;
strip 8: SBA; strip 9: DBA; strip 10: UEA I; strip 11: SJA; strip
12: PNA; strip 13: WGA; strip 14: LCA; strip 15: GSL I; strip 16:
PSA; strip 17: PTL I; strip 18: AAL; strip 19: WGA s+b.
[0121] D: Characterization of the goblet cell antigen after
preparation from an agarose gel
[0122] left-hand track: marker; right-hand track: goblet cell
antigen after preparation from an agarose gel, lectin blot with
PHA-E.
[0123] FIG. 9: Characterization of the glycans of goblet cell
antigen after deglycosylation
[0124] A, B: Lectin blot after deglycosylation with N-glycosidase
F
[0125] left-hand track: sample of the chromatography preparation
after N-glycosidase digestion; right-hand track: sample of the
chromatography preparation without digestion, in each case the same
amounts of the sample.
[0126] A: lane 1: marker; lane 2: N-glycosidase F; strip 1: PHA-L;
strip 2: PHA-E; strip 3: RCA; strip 4: LCA.
[0127] B: lane 1: marker; lane 2: N-glycosidase F; strip 5: PSA;
strip 6: AAL.
EXAMPLES
Example 1
Identification of Goblet Cell-Positive Sera from Colitis Ulcerosa
Patients
[0128] Detection of the antigen to be sought is possible by using
anti-goblet cell-positive sera. Sera from colitis ulcerosa patients
are therefore investigated in immunofluorescence for their
reactivity with goblet cells with the aid of slides with sections
of adult primate intestine, human fetal intestine (from excess
material of pathologically analyzed samples) and human adult
intestine (from surgical resection material). For the investigation
e.g. slides from EUROIMMUN together with associated auxiliary
reagents are used.
[0129] The test is carried out by standard methods, e.g. in
accordance with the instructions for the indirect
immunofluorescence test of EUROIMMUN, wherein conjugates of classes
IgG and IgA are exclusively employed. 200 sera from colitis
ulcerosa patients are analyzed in this manner, and anti-goblet
cell-positive sera can be employed as a positive control for
further experiments.
Results:
[0130] Anti-goblet cell-positive sera show a cloud-like, blurred
fluorescence in a plane above the goblet cells. In addition, a
non-specific background fluorescence can be seen with many sera.
Blood donor sera and samples which react negatively show this
background fluorescence only, with a different intensity depending
on the serum. Sera from colitis ulcerosa patients which react to
goblet cell antigen in this test are referred to as anti-goblet
cell-positive sera.
[0131] In a similar manner to that already demonstrated by Stocker
et al. (1987, ibid), 28% of the sera analyzed were anti-goblet
cell-positive, i.e. they led to the fluorescence typical of goblet
cell antigen.
Example 2
Staining of Goblet Cell Antigen in Intestinal Tissue
[0132] Tissue sections from primate intestine, human fetal and
adult intestine were investigated for goblet cell antigen in
indirect immunofluorescence with anti-goblet cell-positive sera, as
described in Example 1. Expression of further antigens was
investigated.
[0133] For detection of the additionally employed monoclonal
antibodies against the mucins MUC2 (Abcam Ltd) and MUC5AC
(NeoMarkers, Inc.), FITC (fluorescein)-labeled anti-mouse
antibodies of class IgG are employed as conjugates, and for the
anti-MUC3 antibodies (Quartett GmbH) FITC-labeled anti-rabbit
antibodies, also of class IgG, are employed. In the case of double
fluorescence, the slides are washed after serum incubation and then
incubated with one of the monoclonal antibodies for 30 minutes at
room temperature. After the subsequent washing step, a mixture of
anti-human IgG-FITC (diluted 1:5 in PBS-Tween) and anti-mouse
IgG-TRITC (tetramethylrhodamine isothiocyanate) (diluted 1:200 in
PBS-Tween) is employed as conjugate.
Results:
[0134] The fluorescence typical of goblet cell antigen manifests
itself with the anti-goblet cell-positive sera on all the intestine
sections employed (FIGS. 1A and B). The additionally tested
monoclonal antibodies against the mucins 2, 3 and 5AC, which are
all expressed in the intestine (Hayashi et al., 2001, Digestion 63,
28-31) show different patterns and localizations.
[0135] The anti-MUC2 antibody shows a granular fluorescence which
is limited to the cytoplasm of the goblet cells (FIGS. 1C and D).
After incubation with the anti-MUC3 antibody, other intestinal
structures also react, apart from the goblet cells. The anti-MUC5AC
antibody, which is the only one of the monoclonal antibodies used
which reacts with the completely glycosylated antigen, according to
the manufacturer, like the anti-MUC2 antibody shows a goblet
cell-specific fluorescence pattern, the pattern after incubation
with the anti-MUC 5AC antibody being similar to that after the
incubation with goblet cell-positive sera. After incubation with
the anti-MUC2 antibody, however, in addition to a fluorescence
located in the cytoplasm, a membrane-associated fluorescence is
also manifested. The localization of the positive signal can be
illustrated with the aid of double fluorescence with an anti-goblet
cell-positive serum and the goblet cell-specific anti-MUC2
antibody. The anti-MUC2 antibody detected via TRITC-labeled
anti-mouse antibodies reacts--as described--with the cytoplasm of
the goblet cells, and the goblet cell-specific reaction of the
serum rendered visible with FITC-labeled anti-human antibodies is
in the plane above this (FIG. 1B).
Example 3
Fractionation of Intestinal Tissue
[0136] Human adult intestine is weighed in the frozen state and, on
ice, is covered with a layer of about 3 volumes of TNE-T buffer (50
mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF
(phenylmethylsulfonyl fluoride), 0.1% Triton X-100). After thawing,
the tissue is first cut with scissors and then comminuted with a
Miccra D-8 (ART--moderne Labortechnik) three times for 1 minute
each time (level C) while cooling with ice. For cooling the sample,
a pause of 5 minutes is maintained between each step.
[0137] The preparation is centrifuged for 30 minutes at 500.times.g
and 4.degree. C., the supernatant is transferred to a container and
the sediment is resuspended in one volume of TNE buffer (50 mM
Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF). Thereafter, a
further centrifugation step for 30 minutes at 2,000.times.g and
4.degree. C. follows. The supernatant is removed again and the
sediment is resuspended in one volume of TNE buffer.
[0138] The sample is then centrifuged for 30 minutes at
21,250.times.g and 4.degree. C., the supernatant is removed and the
sediment is resuspended in one volume of TNE buffer. If a fatty
layer is visible after this step, the suspension is filtered
through glass wool.
[0139] The preparation is centrifuged for 60 minutes at
100,000.times.g and 4.degree. C. The supernatant is transferred to
a container, the sediment is resuspended in one volume of TNE
buffer and the suspension is then centrifuged for 60 minutes at
220,000.times.g and 4.degree. C. After the supernatant has been
transferred to a container, the sediment is resuspended in one
volume of TNE buffer.
[0140] After the homogenization and subsequent fractionation by
ultracentrifugation, the individual supernatants and samples of the
particular sediments are investigated by gel electrophoresis
(NuPage-System, Invitrogen, using MES buffer) and silver
staining.
[0141] The samples are analyzed in parallel under non-reducing and
reducing (15.6 mM DDT, 1.25% Iodoacetamide) conditions. Marker 12
and MultiMark from Invitrogen are run as markers.
[0142] The silver staining is carried with standard methods, e.g.,
by the method of Heukeshoven (1988, Electrophoresis 9, 28-32).
Results:
[0143] FIG. 2A shows the result of the fractionation. Because of
the high protein concentration in the homogenate, which still
contains all constituents of the intestinal tissue, individual
bands can scarcely be demarcated from one another. The same is true
for the supernatants 1 and 2 and the sample of sediment 1. In
sediment 2, individual bands can already be seen. From the 3rd
centrifugation step, the separating off by the ultracentrifugation
is clearly visible, and individual bands can be clearly demarcated
from one another. In the sample of sediment 5, only few proteins
are still detectable, the molecular weights of these all being
above the 14.4 kDa band of the marker. A removal of individual
tissue constituents is indeed visible, but no striking features can
be seen with respect to the target antigen.
Example 4
Analysis of Fractions in the Neutralization Test
[0144] The dilution at which the corresponding anti-goblet
cell-positive serum just still shows a goblet cell-specific
reaction, determined by a dilution series in immunofluorescence, is
referred to as titer and is employed for a neutralization test with
the samples of the intestine preparation.
[0145] For the neutralization test, various amounts of fractions of
the intestine preparation from Example 3 are added to 100 .mu.l
each of an anti-goblet cell-positive serum (diluted in PBS-Tween,
so that a limiting titer is achieved, see below) and incubated for
30 minutes at room temperature. The preparations are then
centrifuged for 10 minutes at 13,000 rpm in an Eppendorf
centrifuge. The supernatant is employed instead of the serum
dilution for an indirect immunofluorescence with primary human
adult intestinal tissue. A control field which has been incubated
only with the starting dilution of the serum serves as a comparison
here.
Result:
[0146] No neutralizing action of one of the fractions against an
anti-goblet cell-positive serum can be determined in the test.
Since all fields of the slide react with the same intensity and
identical pattern, no conclusion can be made regarding the fraction
in which the target antigen is present. Repeating the test with
increasing concentrations of the individual samples in the
preparation also leads to an identical result.
Example 5
Investigation of Fractions in Western Blot
[0147] The fractions of the intestine preparation are to be
investigated for the presence of the target antigen with the aid of
a specific detection with anti-goblet cell-positive sera in western
blotting. After the separation by SDS gel electrophoresis, the
proteins are transferred onto a nitrocellulose membrane, e.g., with
the wet blotting method by means of a blotting apparatus from the
company Hoefer.
[0148] After preparation, the samples are applied to SDS-PAGE gels,
and, after transfer to nitrocellulose membranes, they are first
stained with Ponceau S and then incubated with anti-goblet
cell-positive or blood donor sera.
[0149] For the Ponceau S staining, the nitrocellulose membrane is
covered with Ponceau S solution for 10 minutes, while shaking.
After the incubation, the membrane is destained again with
distilled water, until the marker and stained proteins are clearly
visible as red bands.
[0150] For the specific immunological detection with antibodies,
the membrane, cut into strips if appropriate, is first blocked for
15 minutes with universal buffer (EUROIMMUN) with 3% (w/v) of
powdered milk.
[0151] It is then incubated for 3 hours with control or patient
sera (1:500 in universal buffer with 3% (w/v) of powdered milk).
Washing is then carried out with universal buffer three times for 5
minutes each time. In a second incubation step, the antibodies
bound to the proteins in the positive case react with a conjugate
solution (EUROIMMUN, diluted 1:10 in universal buffer), which
contains anti-human antibodies labeled with alkaline phosphatase.
Washing is then carried out as after the serum incubation. In a
third incubation step, the bound antibodies are then detected with
an NBT/BCIP substrate solution (4-nitroblue tetrazolium
chloride/chlorobromoindolyl phosphates, EUROIMMUN).
Results:
[0152] After the Ponceau S staining, a similar pattern of bands can
be seen as after silver staining, the intensity of many bands being
weaker because of the lower sensitivity of the Ponceau S staining.
As with the silver gel, no striking features can be seen.
[0153] After the incubation of the membranes with anti-goblet
cell-positive or blood donor sera, signals can be seen on both
western blots (FIGS. 2B and C). In almost all the samples, a band
at approx. 55 kDa reacts particularly clearly, which can also be
detected with the Ponceau S staining, although it has a rather weak
intensity here. However, a comparison of the two membranes shows
that identical patterns of bands result on incubation with
anti-goblet cell-positive sera and sera from blood donors. These
reactions therefore have to be evaluated as non-specific. None of
the striking bands of the Ponceau S staining reacts with the sera
in a detectable intensity. By this test also, no conclusion can be
made regarding the presence of the target antigen in one of the
fractions, since no anti-goblet cell-specific reaction can be
detected.
Example 6
Investigation of the Fractions in Lectin ELISA
[0154] For the lectin ELISA, a MaxiSorb plate from the company Nunc
is coated with streptavidin (100 .mu.l of a 0.5 .mu.g/ml solution
in PBS, overnight at 4.degree. C. or 2 hours at room temperature)
and coated with 100 .mu.l each of lectin dilution (1 .mu.g/ml in
PBS, see Table 1) for 30 minutes at room temperature. The plate is
then washed (three times with 300 .mu.l of wash buffer (EUROIMMUN)
or PBS-Tween each time). An incubation with 100 .mu.l of a dilution
of the fractions of the intestine preparation from Example 3 (1:100
in PBS) per well, respectively, for 30 minutes at room temperature
then follows. After a washing step, blocking with 200 .mu.l each of
0.5% (w/v) BSA in PBS for one hour at room temperature follows.
Thereafter, the plate is washed. For the serum incubation, in each
case 100 .mu.l of serum dilution (1:100 in PBS) are incubated for
30 minutes at room temperature. The plate is washed, before
incubation is carried out with in each case 100 .mu.l of conjugate
solution (EUROIMMUN), which contains peroxidase-labeled anti-human
antibodies, for 30 minutes at room temperature. After another
washing step, the plate is incubated for 10 minutes with 100 .mu.l
of substrate solution (EUROIMMUN) per reaction well at room
temperature. At the end of the 10 minutes, the reaction is stopped
by addition of 100 .mu.l of stop solution (EUROIMMUN) each, and the
extinction at 450 nm is measured with a photometer (Tecan Spectra).
The diagram of a lectin ELISA is shown in FIG. 3.
TABLE-US-00001 TABLE 1 Selection of biotinylated lectins
(Vectorlabs) Lectin Specificity AAL (Aleuria aurantia 1,3-L-fucose
lectin) Con A (concanavalin A) .alpha.-D-glucose .alpha.-D-mannose
DBA (Dolichus biflorus N-acetyl-.alpha.-D-galactosamine agglutinin)
GSL I (Griffonia .alpha.-D-galactose simplicifolia lectin I)
N-acetyl-.alpha.-D-galactosamine HHL (Hippeastrum hybrid
.alpha.-D-mannose lectin) LCA (Lens culinaris .alpha.-D-glucose
.alpha.-D-mannose agglutinin) MAL I (Maackia amurensis
N-acetylneuramic acid-.alpha.-2,3- lectin I) galactose PHA-E
(Phaseolus vulgaris N-acetyl-.alpha.-D-galactosamine
erythroagglutinin) PHA-L (Phaseolus vulgaris
N-acetyl-.alpha.-D-galactosamine leukoagglutinin) PNA (peanut
agglutinin) .beta.-D-galactose PSA (Pisum sativum .alpha.-D-glucose
.alpha.-D-mannose agglutinin) PTL I (Psophocarpus galactosamine
tetragonolobus lectin I) RCA (Ricinus communis .beta.-D-galactose
agglutinin) N-acetyl-.alpha.-D-galactosamine SBA (soya bean
N-acetyl-.alpha.-D-galactosamine agglutinin) .beta.-D-galactose SJA
(Sophora japonica N-acetyl-.alpha.-D-galactosamine agglutinin)
.beta.-D-galactose SNA (Sambucus nigra N-acetylneuramic
acid-.alpha.-2,6- lectin) galactose UEA I (Ulex europaeus
.alpha.-L-fucose agglutinin) WGA (wheat germ
(N-acetyl-.beta.-(1,4)-D-glucosamine).sub.>2 agglutinin) sialic
acids WGA succinylated (wheat N-acetyl-glucosamine germ
agglutinin)
Results:
[0155] Analysis of the reactivity of lectins with the samples of
the intestine preparation leads to different extinction values,
depending on the lectin and fraction. In order to be able to
distinguish between specific and non-specific reactions, the
preparations are incubated in parallel with sera which have tested
anti-goblet cell-positive in the indirect immunofluorescence and
sera from blood donors. In the tests carried out, comparable
extinction values result with each lectin for both preparations
(Table 2). In supernatant 1, e.g., the lectin AAL shows an
increased reactivity which, however, has to be regarded as
non-specific, since anti-goblet cell-positive and blood donor sera
and even the negative control, which was incubated with PBS instead
of serum dilution, react to similar degrees. The lectins RCA, PSA,
LCA and SNA also show increased reactivities, although these are
found both with anti-goblet cell-positive and blood donor sera and
with the control. The reactions therefore have to be rated
non-specific. Comparable results are also achieved in the testing
of the other preparations. No sample of the preparation shows a
specific reactivity with one of the lectins available.
TABLE-US-00002 TABLE 2 Extinction values (450 nm) of a lectin ELISA
with supernatant 1 of the intestine preparation Anti-goblet Lectin
cell-positive sera Blood donor Neg. control UEA I 0.041 0.043 0.032
RCA 0.268 0.182 0.185 Con A 0.170 0.102 0.132 WGA 0.048 0.050 0.039
SBA 0.071 0.031 0.073 DBA 0.030 0.035 0.033 MAL I 0.025 0.037 0.025
PNA 0.028 0.024 0.022 SJA 0.036 0.044 0.022 PHA-L 0.161 0.205 0.160
AAL 0.932 0.895 1.790 LCA 0.571 0.248 0.670 PSA 0.530 0.267 0.643
SNA 0.219 0.116 0.176 HHL 0.082 0.067 0.097 PBS - 0.023 0.037 0.019
control
Example 7
Culture of HT29-18N2 Cells
[0156] The HT29-18N2 cells are a daughter cell line of HT29 cells.
The protocol for the culture and the clone were originally
developed by Prof. Dr. Tom Phillips, University of Missouri,
Columbia (1988, Gastroenterology 94: 1390-1403).
[0157] 1.7.times.10.sup.4 cells/cm.sup.2 are seeded in plastic
bottles and cultured under standard conditions at 37.degree. C.
with 5% CO.sub.2 in Dulbecco's Modified Eagle's medium (DMEM,
Invitrogen) with addition of 10% fetal calf serum (FCS), until
confluence is reached. The cells are then harvested by addition of
trypsin/EDTA and seeded again in DMEgal medium in plastic bottles.
Since the change in the medium composition results in a high death
rate and a slower growth rate, the medium is changed every day.
[0158] DMEgal medium is glucose-free in the sense that it contains
only residues of glucose from the FCS, and these are present in a
lower amount than galactose. It is composed of 8.3 g DME medium
base (Sigma), 0.1% (w/v) galactose (Sigma), phenol red solution (8
mg/ml; Sigma), 0.37% (v/v) NaHCO.sub.3 (Sigma) and 10% (v/v) of
dialyzed FCS (JRH Biosciences).
[0159] When cultured in DMEM, the cells are highly aggregated and
grow in islands in several layers. After seeding in DMEgal, an
increased mortality rate is observed. The surviving cells no longer
grow in islands, but are seen as individual cells, and demarcated
from one another under the microscope.
[0160] When confluence is again reached, frozen cultures of a part
of the cells are prepared in freezing medium (90% (v/v) FCS and 10%
(v/v) DMSO), and are stored in nitrogen.
[0161] The remaining cells are seeded on sterile cover glasses
(26.times.76 mm; 7.2.times.10.sup.6 cells/cover glass). To achieve
a faster growth rate, the DMEgal is replaced by DMEM/10% FCS. After
4 days, the medium is changed from DMEM/10% FCS to DMEgal or
protein-free medium, e.g. Protein Free Hybridoma Medium II (PFHM
II, Invitrogen).
[0162] In parallel, HT29-18N2 cells are cultured in
protein-containing DMEM (DMEM/10% FCS). Unless mentioned otherwise,
the designation HT29-18N2 cells in the following relates to cells
after culture in the protein-free medium PFHM II.
[0163] When a uniform and adequate cell density is reached, slides
are produced from each half of a cover glass using the biochip
technology of EUROIMMUN, and the other half is tested in western
blot after breaking down the cells by means of ultrasound in TNE-T
buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF,
0.1% Triton X-100).
Example 8
Indirect Immunofluorescence with HT29-18N2 Cells
[0164] Fixation with Acetone
[0165] The cover glass is rinsed twice with PBS. Excess liquid is
allowed to drain off on cellulose, before the cover glass is first
rinsed in acetone (Merck) and then fixed in fresh acetone for 10
minutes at room temperature.
Fixation with Formaldehyde
[0166] The cover glass is twice covered with a layer of PBS, and
the PBS is then sucked off and discarded. Residues of liquid are
allowed to drain off on cellulose. The cover glass is then first
covered with a layer of 3.5% formaldehyde (Riedel de Haen) in PBS
(the liquid is removed again as in the preceding step) and then
fixed for 10 minutes with formaldehyde solution at room
temperature.
Fixation with Ethanol
[0167] The procedure is the same as the previous fixation, 100%
ethanol (Merck) being employed instead of the formaldehyde
solution.
Indirect Immunofluorescence
[0168] The fixed slides with cells are stained as described in
Example 1. In contrast to staining of the tissue sections, slides
with cells are rinsed only briefly with PBS/0.2% Tween in a cuvette
during washing, and are then incubated in a further cuvette for 5
minutes in PBS-Tween.
[0169] Slides with differently cultured HT29-18N2 cells and primate
intestine (as a positive control) are then tested with the
anti-goblet cell-positive sera, 288 blood donor sera and a number
of samples from patients with confirmed colitis ulcerosa (57 sera)
or Crohn's disease (80 sera). Incubation is carried out here by the
standard incubation with altered washing steps.
Results:
[0170] The HT29-18N2 cells cultured on cover glasses in DMEgal and
fixed with acetone, formaldehyde or ethanol are tested in indirect
immunofluorescence for their reactivity with goblet cell-positive
sera and sera from blood donors. The use of ethanol and
formaldehyde is only poorly suitable for fixing these cells to
cover glasses. On all the fields of the slide, the cells become
detached during the incubation, so that only a poor conclusion can
be made regarding the reactivity of the cells with the sera used.
Acetone fixing is therefore carried out for all the following
experiments.
[0171] The incubation of cells grown in Protein Free Hybridoma
Medium II (PFHM II) and fixed with acetone at various points in
time with anti-goblet cell-positive sera shows a cloud-like,
blurred fluorescence in a plane above the fixed cells (FIG. 4A).
After incubation of the HT29-18N2 cells with blood donor sera, only
the cells themselves have a weak, uniform fluorescence (FIG.
4B).
[0172] Since only anti-goblet cell-positive sera cause the
cloud-like fluorescence pattern similar to the goblet cell-positive
reaction of fixed intestinal tissue, the reaction is to be regarded
as specific. The longer the cells have grown in the hybridoma
medium, the more intense is the specific signal. After growth in
PFHM II for 4 days, the cells are particularly suitable for
detection of the antigen in indirect immunofluorescence. Cells
grown in DMEM/10% FCS or DMEgal and tested in parallel show no
specific reactivity.
[0173] The 288 sera from blood donors and Crohn's disease patients
tested in the indirect immunofluorescence in parallel on primate
intestine and HT29-18N2 cells show no goblet cell-specific
reactivity. The comparison of the primate intestine and cell line
substrates shows that non-specific background fluorescences, which
can make the diagnosis by means of intestine difficult for some
sera, interferes considerably less with the evaluation of the
cells. In addition, in the case of incubation of sera from patients
with colitis ulcerosa with positive reactions the cloud-like
fluorescence pattern on the cells can be demarcated from the
background considerably better.
[0174] On comparison of the cells grown in various media, a change
in the size and morphology can be seen after changing to PFHM II.
The cells appear to increase in size due to a differentiation, and
the cytoplasm looks considerably more structured.
Example 9
Selectivity of the Binding of Anti-Goblet Cell Antibodies to Goblet
Cell Antigen from HT29-18N2 Cells
[0175] Further antibodies were tested on the cell line in indirect
immunofluorescence. The anti-MUC2 antibody reacts with the
cytoplasm of some cells and therefore also shows a fluorescence
pattern similar to that on intestinal tissue (FIG. 4C). The
anti-MUC3 antibody shows a uniform fluorescence of the cells,
regardless of whether they are grown in DMEM or PFHM II, and
therefore also reacts with the non-differentiated cells. The
reaction of the anti-MUC5AC antibody is limited to the cells grown
in PFHM II. Apart from a granular pattern in many cells, a
fluorescence manifests itself here in a plane above the cells,
which can be distinguished visually from the anti-goblet
cell-specific signal (FIG. 4D).
[0176] Sera from patients with defined diseases, which react with
structures of the HEp-2 cells (human epithelial cells) employed for
autoimmune diagnostics, are tested in order to render conclusions
regarding a demarcation of different fluorescence patterns
possible. These sera contain autoantibodies against cell nuclei
(homogeneous, granular and nucleolar ANA pattern), the nucleus
membrane, mitochondria (AMA), the Golgi apparatus, vimentin and
actin (cytoskeletal structures) and against the antigen Jo-1. These
antibodies react with HT29-18N2 cells, but the arising fluorescence
patterns can be distinguished unambiguously from the reaction with
anti-goblet cell antibodies. These autoantibodies are thus not
cross-reactive with goblet cell antigen.
Example 10
Sensitivity of the Binding of Anti-Goblet Cell Antibodies to Goblet
Cell Antigen from HT29-18N2 Cells
[0177] The sera collections from patients with colitis ulcerosa
still available in this series of experiments (57 sera) are
unsuitable for determining a prevalence of the anti-goblet cell
antibody, since mainly positive sera were removed on the basis of
early study purposes and the collections can therefore no longer be
regarded as representative. Since the sera are pre-characterized
with respect to antibodies against goblet cells, and it was
possible to determine a prevalence of 28% with these samples with
human fetal intestine as the substrate, a comparison can be made
between the fetal intestine, the HT29-18N2 cells and the adult
primate intestine employed.
Results:
[0178] Of the 57 sera from colitis ulcerosa patients still
available, 11 tested positive for antibodies against goblet cells
on fetal intestine (Table 3). These 11 sera also reacted
specifically to goblet cell on the HT29-18N2 cells. Only 8 sera
with antibodies against goblet cells can be determined with the aid
of the adult primate intestine. The marked similarity of the
fluorescence pattern and a comparison of the sensitivity of the
substrates in indirect immunofluorescence therefore show that the
HT29-18N2 cell line is more suitable as a substrate for diagnostics
for detecting anti-goblet cell antibodies than primate
intestine.
TABLE-US-00003 TABLE 3 Comparison of the reactivity of HT29-18N2
cells, human fetal and adult intestine in indirect
immunofluorescence after incubation with patient sera (1:10 in
PBS-Tween). Adult Fetal HT29-18N2 intestine intestine positive 11 8
11 negative 46 49 46
Example 11
Purification of Goblet Cell Antigen
[0179] Since the immunofluorescence shows a reaction in a plane
above the cells in the positive case, the supernatant of the cell
culture is employed for further tests. The medium from the cell
culture flasks of the HT29-18N2 cells in PFHM II is collected, e.g.
via a gas wash bottle, concentrated with a 100 kDa cutoff (Vivaspin
100, VivaScience), exchanged for PBS and tested in ELISA or dot
blot.
[0180] The supernatants obtained from the cell culture are tested
for specific reactions in dot blot. For this, twice, each time 1
.mu.l of the cell culture supernatant (after concentration with a
100 kDa cutoff and exchange of the medium for PBS) are directly
applied to a previously marked position on the nitrocellulose
membrane. In between and thereafter, the membrane is allowed to dry
thoroughly. Subsequent incubation with various anti-goblet
cell-positive and blood donor sera substantially corresponds to a
standard western blot incubation, although the conjugate incubation
is prolonged from 30 minutes to one hour. The success of the
differentiation is checked in parallel with the anti-MUC5AC
antibody, all the cell culture supernatants being positive.
Results:
[0181] The anti-goblet cell-positive sera show different degrees of
reactivity with the individual samples (FIG. 5A). The concentration
of the antigen here seems to depend on the time for which the cells
have grown in PFHM II medium. 2 days after changing the medium from
DMEM to PFHM II, the signal is indeed present, but of weaker
intensity. The longer the cells have grown in PFHM II, the more
intense is the reaction. The anti-goblet cell-positive sera
likewise react to different degrees. The intensity seems to
correlate with the titer in the immunofluorescence.
[0182] The blood donor sera do not react with any of the cell
culture supernatants. The negative control, which was incubated
with universal buffer+3% (w/v) of powdered milk instead of a serum
dilution likewise shows no reactivity.
Example 12
Chromatographic Purification of the Goblet Cell Antigen
[0183] The cell culture supernatants from the cell culture were
separated on the basis of different charges in the molecules with
the aid of anion exchange chromatography. A purification of the
protein mixture contained in the samples is thus to be achieved, in
order to render the development of a test on the basis of the
antigen present in the purest possible form possible (for ELISA,
western blotting).
[0184] The column used is a POROS HQ/M 20 .mu.m from Applied
Biosystems of 16 mm diameter and 10 ml column volume. A BioLogic
Duo Flow from Biorad is used as the HPLC unit. The flow rate is 10
ml/min. Fractions of 5 ml volume are collected.
[0185] The column is first flushed with 50 ml of water. A
regeneration with 50 ml of regeneration buffer (10% acetic acid and
1 M NaCl) then follows. The column is flushed first with 50 ml of
water, thereafter with 10 ml of elution buffer (25 mM Tris-HCl pH
8, 250 mM NaCl, 1 mM PMSF) and then with 80 ml of equilibration
buffer (25 mM Tris-HCl pH 8, 1 mM PMSF), before application of the
sample (5 ml) takes place. The fractions are collected after this
point in time. After application of the sample, 50 ml of
equilibration buffer are passed over the column. The column is
eluted via a gradient which comprises three stages, in each of
which the concentration of the chloride ions on the column is
increased. In this process, 75, 150 and 250 mM NaCl are
employed.
[0186] The collected fractions are analyzed for their reactivity
with anti-goblet cell-positive and blood donor sera by dot blot in
order to be able to make a conclusion regarding the presence of the
target antigen.
Results:
[0187] The anti-goblet cell-positive sera react with the sample
applied (A) and with the last fractions of the chromatography (FIG.
5B). The blood donor sera react neither with the sample applied nor
with one of the fractions. The reaction of the anti-goblet
cell-positive sera is thus to be rated as specific. The negative
control, which was incubated with universal buffer+3% (w/v)
powdered milk instead of a serum dilution, shows no reactivity.
Example 13
Detection of Anti-Goblet Cell-Positive Sera in ELISA with Goblet
Cell Antigen
[0188] By direct coating of an ELISA plate with goblet cell antigen
from cell culture supernatant of HT29-18N2 cells and HT29-18N2
cells (in each case after culture in protein-free medium), the
reactivity of the fractions is to be tested with a mixture of
anti-goblet cell-positive and blood donor sera. The fractions of
the chromatography are furthermore also tested in ELISA in addition
to dot blot.
[0189] The plates are coated overnight at 4.degree. C. or for 2
hours at room temperature with the goblet cell antigen or the cell
lysate. The cell lysate is prepared by freezing and thawing the
cells in TNE-T buffer. The ELISA is further carried out by standard
methods, similar to the lectin ELISA described in Example 6.
Results:
[0190] The preparation with cell lysate only shows a low specific
reaction. The cell culture supernatant tested shows an increased
reactivity with the anti-goblet cell-positive sera, a correlation
being identified between the extinction value and the titer of the
corresponding serum in the indirect immunofluorescence. The sera
with the highest titers determined by immunofluorescence are also
more reactive in the ELISA by comparison, and lead to higher
extinction values. Since the blood donor sera show no comparable
reaction, the signal is to be evaluated as specific. It is clearly
shown in this experiment that the purified goblet cell antigen is
more suitable for a detection in ELISA than the cells themselves
(FIG. 6A).
[0191] The values determined for the fractions of the
chromatographic separation with anti-goblet cell-positive sera in
dot blot can be confirmed in the ELISA (FIG. 6B).
Example 14
Characterization of the Goblet Cell Antigen and Detection in
SDS-PAGE/Western Blot
[0192] The fractions of the anion exchange chromatography which
show a specific reactivity with anti-goblet cell-positive sera in
dot blot are separated by means of SDS-PAGE, the gel
electrophoresis being carried out under reducing (e.g. after
reduction e.g. with mercaptoethanol or dithiothreitol) and
non-reducing conditions. MultiMark is used as the marker. Western
blotting is carried out, wherein anti-goblet cell-positive sera
and, as a control, MUC5AC antibodies are used.
Results:
[0193] Under non-reducing conditions, a band which lies above the
top band of the marker (185 kDa) is detected here on incubation
with anti-goblet cell-positive sera (FIG. 7A). A band which is
above both the top marker band and the band which reacts with the
anti-goblet cell-positive serum is also seen after incubation with
the anti-MUC5AC antibody. The apparent molecular mass (or the
molecular weight) of goblet cell antigen is thus between 185 kDa
and the apparent molecular weight of the non-reduced mucin MUC5AC
(>2,000 kDa). The results furthermore indicate that the
preparation of the goblet cell antigen is contaminated with MUC5AC
after the anion exchange chromatography. This experiment also shows
that western blotting after electrophoretic separation under
non-reducing conditions is a suitable method for detecting
anti-goblet cell antibodies in a sample.
[0194] Under reducing conditions, neither the goblet cell antigen
nor MUC5AC can be detected with the antibodies.
Example 15
Characterization of the Goblet Cell Antigen and Detection in
Agarose Gel Electrophoresis/Western Blot
[0195] Because of the size of the antigen, it is furthermore
analyzed in agarose gel electrophoresis. The optimum separation
range of the method is approx. 100 to 2,000 kDa.
[0196] For the preparation of a gel, 1% (w/v) of agarose and 357 mM
bis-Tris-HCl buffer (pH 6.5) are heated briefly until the agarose
has dissolved completely.
[0197] The mixture is introduced into 10.times.10 cm empty
cassettes (anamed Elektrophorese GmbH), the pocket being shaped
with the aid of a comb (anamed Elektrophorese GmbH). After cooling,
the agarose gel can be employed analogously to the SDS-PAGE method.
The sample preparation is also identical in both cases.
[0198] After the separation of proteins in the gel electrophoresis,
the proteins, as is known for polyacrylamide gels, can be
transferred to a nitrocellulose membrane. The blotting time chosen
here can be somewhat shorter, in the wet blotting method used,
e.g., only 45 minutes.
Results:
[0199] In the separation of the chromatography fractions by means
of agarose gel electrophoresis and subsequent western blotting--as
in the SDS-PAGE--no reactivities of the goblet cell antigen with
the specific antibody can be detected under reducing conditions
(see FIG. 7B). MUC 5AC also can be detected under non-reducing
conditions only. Under non-reducing conditions, in each case only
one band can be seen after incubation with various anti-goblet
cell-positive sera. This is found at the same level for all the
sera. After incubation with the anti-MUC5AC antibody, a colored
region can be seen above the band which occurs after incubation
with anti-goblet cell-positive sera. A band weak in comparison,
which occurs only at a very high concentration of the anti-MUC5AC
antibody, is also to be made out below the latter position. Blood
donor sera show no reactivity.
Example 16
Characterization of the Purity of the Goblet Cell Antigen
Preparation
[0200] The anion exchange chromatography fractions which are
positive for goblet cell antigen are labeled with FITC and
investigated for their purity in agarose gel electrophoresis under
reducing and non-reducing conditions. This is compared to a western
blot of non-labeled fractions. For the labeling, 100 .mu.l of 1 M
NaHCO.sub.3 solution and 2 mg of FITC (Sigma), dissolved in 40
.mu.l of DMSO (Hybaid), are added to 900 .mu.l of sample. This
preparation is incubated overnight at room temperature under
exclusion of light on a shaker (Eppendorf). Thereafter, the
preparation is concentrated with a 10 kDa cutoff (Vivaspin 10,
VivaScience) and washed several times with 1 ml of PBS each time,
until the runnings are colorless.
[0201] The sample can be employed in western blotting after an SDS
gel electrophoresis. The procedure for the western blot incubation
is similar to that of standard western blotting incubation, the
serum incubation being omitted and anti-FITC antibodies labeled
with alkaline phosphatase (Sigma, 1:10,000 in universal buffer)
being employed as the conjugate.
Results:
[0202] With the FITC-labeled samples, individual bands can be seen
under both reducing and non-reducing conditions--in each case in
different positions.
[0203] Under non-reducing conditions, in each case a band is seen
which is at the same level as the band which occurs after
incubation with anti-goblet cell-positive sera and above the band
corresponding to IgM. This band, that is to say the goblet cell
antigen, appears to represent the main protein content of the
preparation. The apparent molecular weight of the goblet cell
antigen seems to lie between that of human IgM (about 950 kDa) and
that of non-reduced MUC5AC (>2,000 kDa).
[0204] In addition, colorations are seen both at the level of the
region stained after incubation with the MUC5AC antibody and at the
level of the band corresponding to the IgM. This result confirms
that the goblet cell antigen preparation is probably contaminated
with MUC5AC. IgM or a protein of similar size could furthermore be
present in the preparation.
[0205] Under reducing conditions, 3 bands are seen after incubation
with the anti-FITC antibody. The top band is at the lower end of
the region stained with the MUC5AC antibody. Two further bands are
seen below the band corresponding to the non-reduced IgM.
Example 17
Characterization of Binding of the Goblet Cell Antigen to
Lectins
[0206] Lectin western blots are carried out after agarose and
polyacrylamide gel electrophoresis under non-reducing and reducing
conditions.
[0207] The staining of western blots with lectins instead of
antibodies or sera is carried out analogously, but universal buffer
(EUROIMMUN) with 3% (w/v) BSA is employed for blocking. The
biotinylated lectins and the corresponding conjugate (ExtrAvidin
alkaline phosphatase conjugates, Sigma), are diluted in universal
buffer (lectins 1:2,000, ExtrAvidin conjugate 1:5,000). The
incubation time is in each case 30 minutes, and for the substrate
incubation 3 minutes are sufficient.
Results:
[0208] In the investigation of non-reduced samples, after agarose
gel electrophoresis and incubation of the blot with the lectins
PHA-L, PHA-E, RCA, Con A, LCA, PSA and AAL (see also Table 1) a
band is seen in each case (FIG. 8A) which is at the same level as
the band which can be seen after incubation with anti-goblet
cell-positive sera. Under the conditions used, the lectins SNA,
HHL, MAL I, SBA, DBA, UEA I, SJA, PNA, WGA, GSL I, PLT I, WGA s+b
do not show any reaction, like the sera from healthy blood
donors.
[0209] In the investigation of reduced samples, an intensive band
is to be seen with the lectins PHA-L, PHA-E, RCA, Con A, LCA, PSA
and AAL, respectively (FIG. 8B), which is at the same level for all
the lectins. A small, less intensely colored band is additionally
visible with these lectins, apart from PHA-L. The lectins SNA, HHL,
MAL I, SBA, DBA, UEA I, SJA, PNA, WGA, GSL I, PLT I, WGA s+b show
no reaction.
[0210] After separation of the reduced samples in SDS-PAGE, three
intensive bands which appear to have a molecular weight of about
56, 66 and 88 kDa can be distinguished with the lectins PHA-L,
PHA-E, RCA, Con A, LCA, PSA and AAL (FIG. 8C). On incubation with
PHA-E, Con A, LCA, PSA and AAL, two bands lying close to one
another are additionally seen in the range of the molecular weights
of 52-54 kDa.
[0211] Since the proteins present in the goblet cell antigen
fraction, apart from the goblet cell antigen itself, are not bound
by the lectins under non-reducing conditions, the proteins detected
under reducing conditions appear to be constituents of the goblet
cell antigen and not constituents of the other proteins.
Example 17
Preparation of the Goblet Cell Antigen from an Agarose Gel and
Analysis of Binding to the Lectin PHA-E
[0212] An agarose gel electrophoresis is carried out as previously
with a non-reduced fraction of the goblet cell antigen after anion
exchange chromatography. The two regions of this gel lying at the
edge underneath the application pocket are separated off by means
of a scalpel. Western blotting with anti-goblet cell-positive serum
is carried out with these two gel fragments in order to determine
the position of the goblet cell antigen in the gel. After the
detection has taken place, the region which contains the goblet
cell antigen is cut out from the remaining agarose gel, incubated
for 10 minutes with NuPAGE sample buffer (Invitrogen) under
reducing conditions and laid in a pocket of a NuPAGE gel. After
running the gel, lectin blotting is carried out as previously with
the lectin PHA-E, which is the most intensely reactive with the
goblet cell antigen.
Result:
[0213] After separation of the reduced sample in SDS-PAGE, three
intensive bands which appear to have an apparent molecular weight
of about 56, 66 and 88 kDa can be distinguished with the lectin
PHA-E (FIG. 8D). The additional two bands lying close to one
another in the regions of the apparent molecular weights of 52-54
kDa are not seen and therefore do not appear to be a constituent of
the goblet cell antigen. The goblet cell antigen thus comprises one
or more peptide chains of 56, 66 and 88 kDa.
Example 18
Characterization of the Glycan Structure of Goblet Cell Antigen
[0214] Sugar residues linked to the protein via an N-glycosidic
bond can be split off with an N-glycosidase. For this, 10 .mu.l of
1% (w/v) SDS are added to 80 .mu.l of the sample and incubation is
carried out for 15 minutes at 37.degree. C. 7.6 .mu.l of 10% (w/v)
Triton X-100 and 4 .mu.l of N-glycosidase F (4 units, Roche AG) are
then added. After incubation at 37.degree. C. overnight, the sample
can be employed in the gel electrophoresis.
Result:
[0215] After the deglycosylation of the samples with N-glycosidase
F, a reactivity with the lectins PHA-L, PHA-E, RCA, LCA, PSA and
AAL is furthermore detected (Con A was not investigated) (FIGS. 9A
and B). Corresponding to the undigested sample, in each case at
least three intensively colored bands are seen which are at the
same level for all the lectins, but which are in each case just
below the corresponding band positions before digestion. Two
correspondingly smaller bands lying close to one another in the
region of the apparent molecular weights of 52-54 kDa are not seen.
The intensity of the reaction has decreased only little due to the
digestion. Both N- and O-glycosidically bonded sugar residues thus
appear to be present in the molecule.
* * * * *